The present invention relates to new synthetic catechol derivatives, in which aromatic azomethine-carboxylic acids, benzhydrazones, amino acids, aminobenzoic acids or dipeptides, pyrrolidine- or oxazolidine-carboxylic acids, or formylcarboxymethyloximes function as structural elements, and relates to conjugates thereof with active ingredients, particularly antibiotics.
It is known that certain catechol structures play an essential role as iron-complexing structural elements in natural siderophores (xe2x80x9cIron Transport in Microbes, Plants and Animalsxe2x80x9d, Eds.: Winkelmann, G., van Helm, D., Neilands, J. B., V. Ch.xe2x80x94Verlagsgesellschaft Weinheim, 1987), e.g. enterobactin, which is a siderophore for E. coli and other bacterial strains, is a trimer of N-(2,3-dihydroxybenzoyl)-L-serine. The monomer is also effective as a siderophore (Hantke, K., FEMS Microbiol. Lett. 67 (1990), 5). N-(2,3-dihydroxybenzoyl)glycine has been found to be a siderophore for B. subtilis (Ito, T., Neilands, J. B., J. Amer. Chem. Soc. 80 (1958), 4645). Some catechol-substituted amino acid derivatives have already been produced synthetically, e.g. N-(2.3-dihydroxy-benzoyl)-L-threonine (Kanai, F., Kaneko, T., Morishima, H., Isshiki, K., Taketa. T., Takeuchi, T., Umezawa, H., J. Antibiot. 38 (1985), 39), N2, N6-bis-(2,3-dihydroxybenzoyl)-L-lysine (Corbin, J. L., Bulen, W. A., Biochemistry 8 (1969), 757; McKee, J. A., Sharma, S. K., Miller, M. J., Bioconjugate Chem. 2 (1991) 281), and N2,N6-bis-(2,3-dihydroxybenzoyl)-lysyl-N6-(2,3-dihydroxybenzoyl)lysine (Chimiak, A., Neilands, J. B., Structure and Bonding 58, (1984), 89). It is also known that certain glyoxylic acid benzhydrazones, oxanilic acid derivatives, etc., can serve as siderophores for different bacterial strains (Reissbrodt, R., Heinishc, L., Mxc3x6llmann, U., Rabsch, W., Ulbricht, H., BioMetals 6 (1993), 155). Some dihydroxybenzylidene-aminobenzoic acids have already been described in the literature, but without any mention of their efficacy as siderophores (Takita, H., Noda, S., Inada. K., Mukaida, Y. S., Toji. M. K., Kobayashi, H., DE 3 414 049 (1984); H. Wolf, Monatsh. Chem. 31 (1910), 903).
Although various catechol compounds have been bonded to xcex2-lactams, by means of which an increase in the antibacterial efficacy of these antibiotics has been achieved due to their transfer into the bacterial cell via bacterial transport routes for iron (e.g. Arisawa, M., Sekine, Y., Shimizu, S., Takano, H., Angehrn, P., Then, R. L., Antimicrob. Agents Chemother. 35 (1991), 653), there is a great need for other new synthetic siderophores with improved pharmacological and pharmaco-kinetic properties, which are suitable for forming conjugates with antibiotics.
On the other hand, as chelating agents for iron, siderophores are potentially capable of influencing the biological metabolism of iron, and diseases associated therewith, in various ways. Thus the siderophore desferrioxamine (desferal) is successfully used in diseases which are caused by an excess of iron (e.g. thalassaemia).
The underlying object of the present invention is to discover new synthetic catechol derivatives which comprise aromatic azomethine-carboxylic acids, benzhydrazones, amino acids, aminobenzoic acids or dipeptides, pyrrolidine- or oxazolidine-carboxylic acids, and formylcarboxymethyloximes as basic structures, which can function as siderophores and/or as biological chelating agents for iron, and which in the form of their conjugates with active ingredients, e.g. antibiotics, effect improved penetration of these compounds into bacterial cells and thereby increase the antibacterial efficacy thereof, and which make it possible to combat penetration-related resistance to antibiotics in bacterial infections in an improved manner.
The compounds according to the invention are effective as siderophores for gram-negative bacteria, i.e. they can supply bacteria with iron ions, and, in die form of their conjugates with active ingredients, e.g. with antibiotics (as xe2x80x9csiderophore-antibiotic conjugatesxe2x80x9d), can transfer these compounds into the bacterial cell via iron transport routes and can thereby improve or even extend the efficacy thereof.
Moreover, the compounds according to the invention are more effective and can be produced more easily than previously known compounds, and in die form of their conjugates with active ingredients make it possible to combat penetration-related resistance to antibiotics in bacterial infections in an improved manner. Furthermore, the present invention provides new chelating agents for iron, which can influence the biological metabolism of iron and which can thus influence diseases associated therewith in various ways.
New synthetic catechol derivatives are provided, of general formula I 
wherein the R1 radicals are identical to or independent of each other and denote OH and/or Oacyl, and R2 represents the following groups in the 3- and/or 4-position:
a. aromatic azomethine-carboxylic acid residues and/or azobenzene-carboxylic acid residues: 
X=CM, N, CH=CH-CH
Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group,
R3=one or two Oacyl radicals when R1=OH or Oacyl, or
R3=H when R1=Oacyl, or 
R15=radicals which, identically to or independently of each other, represent H and/or Oacyl, or 
Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group,
R3=radicals which, identically to or independently of each other, denote H, OH, Oacyl,
b. benzhydrazone radicals: 
R15=radicals which, identically to or independently of each other, denote H, OH, Oacyl,
R4 and/or R5 is H or COY, wherein
Y=OA, with A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group,
c. aminobenzoic acid residues 
Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group,
R19=H, alkyl,
R20=H, alkyl, halogen, OH, Oalkyl, Oacyl, or 
R19 and R21, identically to or independently of each other, each denote H, OH, Oacyl or Oalkyl in the 2,3- and/or 3,4-position
d. amino acid residues: 
Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group,
R6=alkyl, hydroxyalkyl (comprising C1-C5 when R1=Oacyl and C3-C5 when R1=OH), or alkoxyalkyl, acyloxyalkyl, arylalkoxyalkyl, or 
R15 represents, identically to or independently of each other, H, OH, Oacyl,
n is an integer between 1 and 5 when R1 is Oacyl and R15 is H and/or Oacyl, or
n is an integer between 1 and 3 when R1 is OH and R15 is H and/or OH, or 
R15=radicals which, identically to or independently of each other, denote H, OH, Oacyl, n1 and n2 represent an integer between 1 and 5,
e. pyrrolidine- and/or oxazolidine-carboxylic acid residues 
Z=O, CH2,
R16 and R17, independently of each other, denote H, alkyl or aryl,
Y=OA where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an NH or OH group,
f. formyl-O-carboxymethyloximes
R2=CHxe2x95x90NOCH2COY, where
Y=OA, where A=H, alkyl, aryl, aralkyl, an alkali metal ion (preferably Na, K), an ammonium ion or a substituted ammonium ion, or
Y=an active ingredient residue which contains an OH or NH group.
In the above formulae and hereinafter, the term xe2x80x9cacylxe2x80x9d denotes a straight-chain or branched C1-C6 alkanoyl or a straight-chain or branched C1-C6 alkoxy-carbonyl. A straight-chain or branched alkyl and a straight-chain or branched alkoxy, also in compound words such as alkoxyalkyl or acyloxyalkyl, denote a straight-chain or branched C1-C8 alkyl or -alkoxy in particular. Aryl denotes phenyl and substituted phenyl in particular, such as a phenyl which is substituted by a straight-chain or branched alkyl, by a halogen, particularly Cl or F, by a straight-chain or branched alkoxy, hydroxy or carboxy, or by a straight-chain or branched alkoxycarbonyl, by a halogen-substituted alkyl or a substituted phenyl, and aralkyl denotes phenylmethyl and 1- or 2-phenylethyl in particular. The cited radicals R3, R5, R15, R20 and COY may be situated in all possible positions. A substituted ammonium ion is an ammonium ion which is substituted by an alkyl, for example.
The term xe2x80x9cactive ingredient residuexe2x80x9d denotes the residue of any suitable antibacterial active ingredient comprising a free NH or OH group, for example, wherein the active ingredient is esterified or converted to an amide with the catechol radical via this NH or OH group. The bond between the catechol derivative and the antibiotic can be formed either directly or via customary linker groups, e.g. aminocarboxylic acids, hydroxycarboxylic acids, diamines or diols. The term xe2x80x9cantibioticxe2x80x9d is to be understood, for example, as a corresponding xcex2-lactam containing an NH or OH group, e.g. a cephalosporin, e.g. cephalexin, cephadroxil or claforan, or a penicillin, e.g. ampicillin or amoxicillin, or a tetracycline derivative, e.g. an aminodioxycycline, or an antibiotic of die aminoglycoside, macrolide, quinolone or carbapenem type.
If asymmetric C atoms are present, the invention likewise relates to the corresponding D- and L- forms, enantiomers and diastereomers, and to racemates and mixtures of enantiomers and diastereomers.
The compounds according to the invention can be prepared for example, by
a. the reaction of catechol-substituted benzaldehydes (formula I, where R2=CHO), in a suitable solvent such as ethanol or toluene, with a water trap or with water-bonding means such as a molecular sieve in a soxhlet attachment, at reaction temperatures between +50xc2x0 C. and +120xc2x0 C. and generally at the boiling point of the solvent, with corresponding aminobenzoic acids to form aromatic azomethine-carboxylic acids (formula I, where R2=R7 or R8),
or by
b. the reaction of catechol-substituted benzhydrazides (formula I, where R2=CONHNH2), in a suitable solvent such as water, ethanol or acetic acid, at temperatures between +10xc2x0 C. and +120xc2x0 C. and preferably at the boiling point of the solvent, with corresponding formylbenzoic acids or with phenylglyoxylic acids to form corresponding benzhydrazones (formula I, where R2=R9),
or by
c. the reaction of di(acyloxy)benzoyl chlorides (formula I, where R1=OCOCH3 and R2=COCl, for example) with aminobenzoic acids or esters thereof, in a suitable solvent such as tetrahydrofuran together with a tertiary amine e.g. triethylamine, at a temperature between xe2x88x9230xc2x0 C. and +20xc2x0 C., or in aqueous sodium bicarbonate solution at 0xc2x0 C. to 10xc2x0 C. to form N-[di(acyloxy)benzoyl] aminobenzoic acids or esters, and the last-mentioned esters are optionally converted into the free acids (formula I, where R2=R10),
or by
d. the reaction of 2,3-di(benzyloxy)benzoyl chloride (formula I, where R1=OCH2C6H5 and R2=COCl), in a suitable solvent such as tetrahydrofuran together with a tertiary amine e.g. triethylamine, at a temperature between xe2x88x9230xc2x0 C. and +20xc2x0 C., or in aqueous sodium bicarbonate solution at 0xc2x0 C. to +10xc2x0 C., with amino acids, diamino acids or dipeptides, to form the corresponding, protected N-[2,3-di(benzyloxy)-benzoyl]-amino acids, and die latter are then converted, by customary methods of removing the protective groups, for example by catalytic hydrogenation in ethanol, into the free catechol-substituted amino acid derivatives or peptide derivatives (formula I, where R2=R11, R12, R13 or R14, wherein Z=CH2),
or by
e. the reaction of dihydroxy- or diacyloxybenzoyl chloride (formula I, where R1=OH or Oacyl and R2=COCl) with an oxazolidine carboxylate, obtained by known methods from serine and aldehydes, for example formaldehyde, in aqueous alkaline solution, with subsequent acidification in a suitable solvent, for example in ethanol or in an ethanol/water mixture, at a temperature betweenxe2x80x9410xc2x0 C. and +10xc2x0 C., to form substituted oxazolidine,-carboxylic acid derivatives (formula I, where R2=R14 and Z=O),
or by
f. the reaction of catechol-substituted benzaldehyde (formula I, where R2=CHO), in a suitable solvent, with O-carboxymethylhydroxylamine or salts thereof to form the corresponding formyl-O-carboxymethyloximes (formula I, where R2=CH=NOCH2COOH).
The compounds of formula I according to the invention in which Y in R2=an active ingredient residue which comprises a free NH or OH group are prepared, for example, by the reaction of a compound of formula I in which Y in R2=OH, e.g. by the mixed anhydride method, firstly with chloroformic acid ester and a tertiary amine, e.g. triethylamine, and then with the corresponding active ingredient which contains a free NH or OH group and which optionally contains a customary linker group, such as residues of a diamino carboxylic acid, of a hydroxycarboxylic acid or of a diamine or diol, together with a suitable tertiary amine, e.g. triethylamine, in a suitable solvent, e.g. tetrahydrofuran.
The compounds of formula I which contain a carboxyl group may exist as free acids, in the form of their salts or as readily cleavable esters, particularly esters which can be cleaved under physiological conditions. The compounds are purified by the usual methods known from the prior art, for example by recrystallisation or by means of chromatographic methods.
The compounds according to the invention are effective as siderophores for various gram-negative bacterial strains.
Testing for siderophore efficacy was performed using various bacterial indicator mutants which only exhibit reduced growth due to lack of siderophores and which are capable of an increase in growth after the addition of the test substances as substitute siderophores. In the indicator mutants, the synthesis of the respective siderophores, e.g. pyoverdin, pyochelin, enterobactin, aerobactin or yersiniabactin, or the biosynthesis of aromatic compounds, is blocked, or there is a lack of receptors for enterobactin, pyochelin or pyoverdin and for other important components for the bacterial transport of iron (e.g. the membrane proteins Cir, Fiu, FepA and TonB). Under conditions where there is a lack of iron, these mutants therefore cannot grow or can only grow to a very slight extent. In particular, the following indicator mutants were used: Pseudomonas aeruginosa PAO 6609, K 407, 690; E. coli AB 2847, Salmonella typhimurium enb-7, TA 270; Klebsiella pneumoniae KN 4401; Yersinia enterocolitica WAH; Proteus mirabilis 12 (wild); Proteus vulgaris 718 (wild) and Morganella morganii SBK3 (wild). The wild strains denoted by xe2x80x9cwildxe2x80x9d only possess iron absorption systems which are inadequate, which is why the addition of a siderophore results in increased growth. The controls used were ferrioxamine E for the Pseudomonas strains, ferrioxamine G and enterobactin for the Salmonella strains, ferrichrome for the E. coli, Klebsiella and Y. enterocol. strains, and 3,4-dihydroxybenzylidene-2,4,6-trimethylaniline for Morganella morganii (see the above literature reference by R. Reissbrodt et al.).
For the E. coli mutants IR 112 and H 1728 lacking the membrane proteins TonB or Cir and Fiu, which are important for active iron transport, all the substances tested had no effect. This is an indication that the substances act purely as siderophores.
The growth areas of the indicator mutants (diameter in mm) under the effect of the test substances are given in Tables 1-3. The annotations + and (+) relate to non-specific promotion of growth.
Due to their properties as bacterial siderophores, the compounds of general formula I can serve as transport vehicles or penetration accelerators for antimicrobial antibiotics and other active ingredients, i.e. in conjugates with antibiotics or other active ingredients they can serve to transport the latter into the microbial cell via iron transport routes and can thus increase their efficacy.
Compounds of general formula I, where Y in R2=an active ingredient residue, possess an antibacterial efficacy, for example even in part against bacteria which are resistant to other xcex2-lactams. Therefore, a few compounds of general formula I, where Y=an active ingredient residue, e.g. substances 28-37, were tested in an agar diffusion test against particular bacterial strains which are in part resistant to other xcex2-lactams (Table 4). The following strains were used: Pseudomonas aeruginosa SG 137 (carbenicillin-resistant), KW 799 WT (wild type), KW 799/61 (penetration mutant, cell wall damaged, penetration made easier), ATCC 27853 (wild type), ATCC 9027 (wild type), NCTC 10662 (ATCC 25668, clinical isolate, carbenicillin-sensitive), NCTC 10701 (carbenicillin-sensitive), NPS1 and Oxa6 (plasmid-coded xcex2-lactamase); E. coli DCO (wild type), DC2 (penetration mutant, cell wall damaged, penetration made easier), Klebsiella pneumoniae ATCC 10031 (wild type), as well as SG 117; Salmonella gallinarum ATCC 9184; Stenotrophomonas maltophilia GN 12873 (ampicillin-, azlocillin- and carbapenem-resistant), and IMET 10402.
Surprisingly, it was found that the substances tested exhibited outstanding efficacy, not only for ampicillin-resistant and/or xcex2-lactamase inhibitor-resistant wild type strains, but that they were also effective for two Pseudomonas strains comprising plasmid-coded xcex2-lactamase (NPS1, Oxa6) and multi-resistant Stenotrophomonas strains, whilst azlocillin, and in part meropenem and imipenem also, for example, were ineffective.
The surprisingly good efficacy was also verified in a microdilution test. The minimum inhibiting concentrations (MICs) were determined for the following bacterial strains: Pseudomonas aeruginosa NCTC 10701, NCTC 10662, SG 137, ATCC 27853, KW 799 WT and KW 799/61, E. coli DCO, DC2 and ATCC 25922, Serratia marcescens SG 621; Salmonella gallinarum ATCC 9184, Klebsiella pneumoniae ATCC 10031 and SG 117.
The results of the tests are given in Table 5. According to these results, all the siderophore-ampicillin conjugates were highly effective compared with azlocillin and ampicillin as the standards, particularly against Pseudomonas aeruginosa SG 137, which is a germ which is particularly resistant to carbenicillin. They were also highly effective against wild type strains of Pseudomonas, and were also effective in part against E. coli and Serratia.
With the test germs KW 799/WT and /61 of Pseudomonas and DCO and DC2 of E. coli, the effect of improved penetration capacity on the efficacy of the substances was investigated. KW 799/61 and DC2 are mutants which possess a more penetrable outer membrane compared with the wild types KW 799/WT and DCO, respectively. For the comparison substances azlocillin and ampicillin, penetration capacities which were poor to a greater or lesser extent were determined from the considerable differences in their activity against the wild type and against mutants. This was in contrast to the behaviour of the conjugates, which exhibited a good penetration capacity.
The results obtained with penetration mutants of Pseudomonas, KW 799/61, and of E. coil DC2 and wild types thereof confirmed that most of the new substances possessed a considerably better penetration capacity than ampicillin and azlocillin. By means of further experiments using special E. coli mutants which lack the porins ompC and ompF, via which xcex2-lactams normally enter the bacterial cell, or which lack the membrane protein tonB, which is essential for the active transport of iron, it was shown that the siderophore-antibiotic conjugates described above are capable of utilising two penetration paths (via the porins ompC and ompF and via the tonB iron transport path), whilst the antibiotic activity of ampicillin and azlocillin depended only on the presence of the porins. The efficacy against xcex2-lactamase formers and against multi-resistant germs is therefore due to a new type of mechanism for overcoming the penetration resistance, by means of which the ratio of active ingredient to enzyme in the bacterial cell is influenced so that not all antibiotic molecules are inactivated before they reach their target.
PLB268: ompF- was super-expressed.
Furthermore, the results of a CAS test are given in Table 7. The CAS test (chromazurol-S test) of Schwyn and Neilands (Anal. Biochem. 160, 47 (1987)) is based on a colour reaction due to the dissolution leaching of Fe from the chromazurol-S complex and its binding by the catechol compound, by means of which the property of the compound as a siderophore is detected. The CAS tests were positive for the new substances, whilst they were completely negative for ampicillin and azlocillin. This also verifies the surprising discovery that the new antibiotics enter the bacterial cell in an enhanced manner, namely via an iron transport route in addition to the porin route.
On account of their properties as siderophores or as chelating agents for iron, compounds of general formula I, and also the salts thereof when acidic groups are present, and also the esters thereof which can cleave under physiological conditions, are suitable for application as drugs for diseases which are caused by a disorder of the physiological metabolism of iron. On account of their antibacterial efficacy, compounds of general formula I in which Y in R2=an active ingredient residue, e.g. the residue of an antibiotic containing an NH or OH group, and also the salts thereof when acidic groups arc present, and the esters thereof which can cleave under physiological conditions, are suitable as drugs for combatting bacterial infections in humans and working animals.
Compounds of formula I can be used for said diseases either on their own or in the form of pharmaceutical preparations with physiologically compatible adjuvant or carrier materials which are known in the art, wherein all customary pharmacological forms of application are possible in principle.